CpxR Binding to Promoter Regions

The expression vector pET-28a-cpxR was constructed, and the recombinant protein CpxR was obtained and phosphorylated according to a previously reported procedure (12 (link)). The promoter regions of the apfA and pilM genes were amplified by PCR and labeled by terminal deoxynucleotidyl transferase (TdT) using an EMSA probe biotin labeling kit (Beyotime, China), generating biotin-labeled probes. The binding of His-CpxR to the biotin-labeled probes was performed using a chemiluminescent EMSA kit (Beyotime) according to the procedure recommended by the manufacturer. Samples were loaded onto a 4% nondenaturing polyacrylamide gel buffered with 0.5× Tris-borate-EDTA (TBE) and blotted onto nylon membrane (Beyotime). Visualization was performed using the chemiluminescent EMSA kit (Beyotime).